Helical structure motifs made searchable for functional peptide design

The systematic design of functional peptides has technological and therapeutic applications. However, there is a need for pattern-based search engines that help locate desired functional motifs in primary sequences regardless of their evolutionary conservation. Existing databases such as The Protein Secondary Structure database (PSS) no longer serves the community, while the Dictionary of Protein Secondary Structure (DSSP) annotates the secondary structures when tertiary structures of proteins are provided. Here, we extract 1.7 million helices from the PDB and compile them into a database (Therapeutic Peptide Design database; TP-DB) that allows queries of compounded patterns to facilitate the identification of sequence motifs of helical structures. We show how TP-DB helps us identify a known purification-tag-specific antibody that can be repurposed into a diagnostic kit for Helicobacter pylori. We also show how the database can be used to design a new antimicrobial peptide that shows better Candida albicans clearance and lower hemolysis than its template homologs. Finally, we demonstrate how TP-DB can suggest point mutations in helical peptide blockers to prevent a targeted tumorigenic protein-protein interaction. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/.

Structural Changes of β-Casein Induced by Temperature and pH Analysed by Nuclear Magnetic Resonance, Fourier-Transform Infrared Spectroscopy, and Chemometrics

This study investigated structural changes in β-casein as a function of temperature (4 and 20 °C) and pH (5.9 and 7.0). For this purpose, nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy were used, in conjunction with chemometric analysis. Both temperature and pH had strongly affected the secondary structure of β-casein, with most affected regions involving random coils and α-helical structures. The α-helical structures showed great pH sensitivity by decreasing at 20 °C and diminishing completely at 4 °C when pH was increased from 5.9 to 7.0. The decrease in α-helix was likely related to the greater presence of random coils at pH 7.0, which was not observed at pH 5.9 at either temperature. The changes in secondary structure components were linked to decreased hydrophobic interactions at lower temperature and increasing pH. The most prominent change of the α-helix took place when the pH was adjusted to 7.0 and the temperature set at 4 °C, which confirms the disruption of the hydrogen bonds and weakening of hydrophobic interactions in the system. The findings can assist in establishing the structural behaviour of the β-casein under conditions that apply as important for solubility and production of β-casein.

Design, synthesis, and structural characterization of helix-forming aliphatic homo-δ-peptides based on conformational restriction due to the structural characteristics of cyclopropane

Considerable effort has been directed toward developing artificial peptide-based oligomers that fold into a specific secondary structure, i.e., peptide foldamers. To date, however, detailed structural analysis of crystals of δ-peptide foldamers consisting of aliphatic δ-amino acids, which have a more extended carbon backbone compared with well-studied β- and γ-amino acids, have not been reported. We rationally designed aliphatic homo-δ-peptide foldamers forming a stable helical structure utilizing a chiral cyclopropane δ-amino acid as a monomer unit whose conformation was tightly restricted by the structural characteristics of cyclopropane depending on its stereochemistry. We stereoselectively synthesized the cyclopropane δ-amino acid monomer and prepared its various homo-oligomers. Structural analysis of the homo-δ-peptides using nuclear magnetic resonance, circular dichroism, and infrared spectroscopy revealed that they form a stable 14-helical structure in solution. Furthermore, the effective conformational regulation of the backbone due to the characteristics of cyclopropane allowed us to achieve X-ray crystallographic analysis of the homo-δ-peptides, showing their common right-handed 14-helical structures. The helical structures were consistent with both those predicted by theoretical calculations and those obtained based on nuclear magnetic resonance spectroscopy in solution. A critical point is that the helical structures of these δ-peptides are theoretically predictable by calculations. To our knowledge, this is the first example of aliphatic homo-δ-peptide foldamers forming a stable helical structure both in solution and in crystal.

3D Printed Buccal Films for Prolonged-Release of Propranolol Hydrochloride: Development, Characterization and Bioavailability Prediction

Gelatin-polyvinylpyrrolidone (PVP) and gelatin-poly(vinyl alcohol) (PVA) mucoadhesive buccal films loaded with propranolol hydrochloride (PRH) were prepared by semi-solid extrusion 3D printing. The aim of this study was to evaluate the effects of the synthetic polymers PVP and PVA on thermal and mechanical properties and drug release profiles of gelatin-based films. The Fourier-transform infrared spectroscopy showed that hydrogen bonding between gelatin and PVP formed during printing. In the other blend, neither the esterification of PVA nor gelatin occurred. Differential scanning calorimetry revealed the presence of partial helical structures. In line with these results, the mechanical properties and drug release profiles were different for each blend. Formulation with gelatin-PVP and PRH showed higher tensile strength, hardness, and adhesive strength but slower drug release than formulation with gelatin-PVA and PRH. The in silico population simulations indicated increased drug bioavailability and decreased inter-individual variations in the resulting pharmacokinetic profiles compared to immediate-release tablets. Moreover, the simulation results suggested that reduced PRH daily dosing can be achieved with prolonged-release buccal films, which improves patient compliance.

Insights into Paramyxovirus Nucleocapsids from Diverse Assemblies

All paramyxoviruses, which include the mumps virus, measles virus, Nipah virus, Newcastle disease virus, and Sendai virus, have non-segmented single-stranded negative-sense RNA genomes. These RNA genomes are enwrapped throughout the viral life cycle by nucleoproteins, forming helical nucleocapsids. In addition to these helical structures, recombinant paramyxovirus nucleocapsids may occur in other assembly forms such as rings, clam-shaped structures, and double-headed nucleocapsids; the latter two are composed of two single-stranded helices packed in a back-to-back pattern. In all of these assemblies, the neighboring nucleoprotein protomers adopt the same domain-swapping mode via the N-terminal arm, C-terminal arm, and recently disclosed N-hole. An intrinsically disordered region in the C-terminal domain of the nucleoproteins, called the N-tail, plays an unexpected role in regulating the transition among the different assembly forms that occurs with other viral proteins, especially phosphoprotein. These structures, together with the helical nucleocapsids, significantly enrich the structural diversity of the paramyxovirus nucleocapsids and help explain the functions of these diverse assemblies, including RNA genome protection, transcription, and replication, as well as encapsulation.

Non-Canonical Helical Structure of Nucleic Acids Containing Base-Modified Nucleotides

Chemically modified nucleobases are thought to be important for therapeutic purposes as well as diagnosing genetic diseases and have been widely involved in research fields such as molecular biology and biochemical studies. Many artificially modified nucleobases, such as methyl, halogen, and aryl modifications of purines at the C8 position and pyrimidines at the C5 position, are widely studied for their biological functions. DNA containing these modified nucleobases can form non-canonical helical structures such as Z-DNA, G-quadruplex, i-motif, and triplex. This review summarizes the synthesis of chemically modified nucleotides: (i) methylation, bromination, and arylation of purine at the C8 position and (ii) methylation, bromination, and arylation of pyrimidine at the C5 position. Additionally, we introduce the non-canonical structures of nucleic acids containing these modifications.

Solid-State NMR Studies of the Succinate-Acetate Permease from Citrobacter Koseri in Liposomes and Native Nanodiscs

The succinate-acetate permease (SatP) is an anion channel with six transmembrane domains. It forms different oligomers, especially hexamers in the detergent as well as in the membrane. Solid-state NMR studies of SatP were carried out successfully on SatP complexes by reconstructing the protein into liposomes or retaining the protein in the native membrane of E. Coli., where it was expressed. The comparison of 13C-13C 2D correlation spectra between the two samples showed great similarity, opening the possibility to further study the acetate transport mechanism of SatP in its native membrane environment. Solid-state NMR studies also revealed small chemical shift differences of SatP in the two different membrane systems, indicating the importance of the lipid environment in determining the membrane protein structures and dynamics. Combining different 2D SSNMR spectra, chemical shift assignments were made on some sites, consistent with the helical structures in the transmembrane domains. In the end, we pointed out the limitation in the sensitivity for membrane proteins with such a size, and also indicated possible ways to overcome it.